Abstract There is a need to develop diabetes drugs with a mechanism of action distinct from that of established agents. Defects in adipocyte function drive the onset of systemic insulin resistance and obesity-linked diabetes. The ability of hypertrophied adipocytes to dispose of glucose and lipids and secrete insulin-sensitizing adipokines is severely compromised and this contributes to hyperglycemia, hyperlipidemia, insulin resistance, inflammation, and lipid deposition in tissues such as liver where it dampens insulin action. Agents that can revert these defects and restore natural lipid partitioning amongst tissues are useful insulin sensitizers in humans. The critical challenge that this project addresses is the identification and therapeutic validation of new molecular pathways that can be pharmacologically modulated to revert adipocyte defects and restore insulin signaling in liver and other tissues. Our multidisciplinary team will achieve this goal by combining cutting-edge phenotypic screening and chemoproteomic technologies, with deep expertise in adipose tissue and liver biology and lipid metabolism. We have developed innovative strategies that integrate phenotypic screening with chemoproteomics to streamline the identification of protein targets of bioactive small molecules, which we initially applied to enzymes of the serine hydrolase family, but have more recently extended for proteome-wide investigations. We propose to complement our initial serine hydrolase-focused approach with our new powerful platforms for integrated phenotypic screening and chemical biology to enable the rapid, systematic, and proteome-wide discovery of metabolism targets. By screening unique libraries of small-molecules for desirable phenotypes in adipocytes and hepatocytes in an unbiased manner, we will identify in tandem physiologically relevant proteins and chemical tools to perturb the function of these proteins to expedite their functional annotation and therapeutic validation in diabetes and NASH. In the process, we will create first-in-class chemical probes and genetic models to study key metabolic pathways that will be distributed to the larger research community. Our cutting-edge chemical biology platforms radically expand the portion of the proteome that can be targeted with small molecules. Application of these tools to the central problem that drives diabetes endows this project with unparalleled potential to discover new targets to treat diabetes and associated conditions.